GS-FLX SAMPLE SUBMISSION FORM (PDF)

1. Sample Input
The Roche GS-FLX^TM genome sequencer system supports the sequencing of samples from a variety of starting materials, including genomic DNA, PCR products, BACs, and cDNA.

Genomic DNA
Sample requirements are 10 µg of high molecular weight genomic DNA with supporting Quality Control (QC) documentation (Gel Image).

PCR Products
Please enquire as specific amplification steps are required before the sample is submitted.

cDNA
HMW (>500bp) 15 µg of cDNA.
LMW (70-500bp) 4 µg of cDNA.
We require supporting QC documentation (Gel image or Bioanalyser DNA chip image if possible).

BACs
Please enquire as the DNA has to be prepped with specific protocols.

2. Sample Fragmentation
Samples such as genomic DNA and BACs are fractionated into small 300 to 800 base-pair fragments. For some samples, such as small non-coding RNA, fragmentation is not required. Short PCR products can be amplified using genome sequencer fusion primers to go directly to Step 4, shown below.

3. Adaptor Ligation
Using a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - are added to each fragment. The adaptors will also be used for purification, amplification, and sequencing steps. Single-stranded fragments are used in subsequent steps in the workflow.

4. One Fragment = One Bead
The adaptors enable hundreds of thousands of single-stranded fragments to bind to their own unique beads. The beads are then encapsulated into individual droplets formed by a water-in-oil emulsion, creating a microreactor containing one bead with one unique fragment. Each unique fragment is amplified without the introduction of competing or contaminating sequences. The entire fragment collection is amplified in parallel.

5. One Bead = One Read
The emPCR process amplifies each fragment to a copy number of several million per bead. Subsequently, the emulsion is broken while the fragments remain bound to their specific beads. After enrichment, the clonally amplified bead is ready to load onto the PicoTiterPlate device for sequencing.

6. Sequence Generation and Data-Analysis Tools
The Roche GS-FLX^TM system produces over 1 000 000 reads per 10-hour instrument run. For sequencing-data analysis please inquire.